This invention relates to new replication competent adenovirus vectors comprising an internal ribosome entry site which replicate preferentially in target cells. The present invention also relates to cell transduction using adenovirus vectors comprising an internal ribosome entry site.
Diseases involving altered cell proliferation, particularly hyperproliferation, constitute an important health problem. For example, despite numerous advances in medical research, cancer remains the second leading cause of death in the United States. In the industrialized nations, roughly one in five persons will die of cancer. Traditional modes of clinical care, such as surgical resection, radiotherapy and chemotherapy, have a significant failure rate, especially for solid tumors. Neoplasia resulting in benign tumors can usually be completely cured by surgical removal of the tumor mass. If a tumor becomes malignant, as manifested by invasion of surrounding tissue, it becomes much more difficult to eradicate. Once a malignant tumor metastasizes, it is much less likely to be eradicated.
Excluding basal cell carcinoma, there are over one million new cases of cancer per year in the United States alone, and cancer accounts for over one half million deaths per year in this country. In the world as a whole, the five most common cancers are those of lung, stomach, breast, colon/rectum, and uterine cervix, and the total number of new cases per year is over 6 million.
In the United States, transitional cell carcinoma (TCC) accounts for 90 to 95 percent of all tumors of the bladder. Squamous cell carcinoma (SCC) represents 5 to 10 percent, and adenocarcinoma approximately 1 to 2 percent. Squamous cell and adenomatous elements are often found in association with transitional cell tumors, especially with high grade tumors. Bladder cancer is generally divided into superficial and invasive disease. A critical factor is the distinction between those tumors that are confined to the mucosa and those that have penetrated the basement membrane and extended into the lamina propria. The term xe2x80x9csuperficial bladder tumorxe2x80x9d is generally used to represent a tumor that has not invaded the muscularis. Invasive tumors are described as those that have invaded the muscularis propria, the perivesical fibroadipose tissue, or adjacent structures. Carcinoma in situ (CIS) is a high grade and aggressive manifestation of TCC of the bladder that has a highly variable course.
A number of urothelial cell-specific proteins have been described, among which are the uroplakins. Uroplakins (UP), including UPIa and UPIb (27 and 28 kDa, respectively), UPII (15 kDa), and UPIII (47 kDa), are members of a group of integral membrane proteins that are major proteins of urothelial plaques. These plaques cover a large portion of the apical surface of mammalian urothelium and may play a role as a permeability barrier and/or as a physical stabilizer of the urothelial apical surface. Wu et al. (1994) J. Biol. Chem. 269:13716-13724. UPs are bladder-specific proteins, and are expressed on a significant proportion of urothelial-derived tumors, including about 88% of transitional cell carcinomas. Moll et al. (1995) Am. J. Pathol. 147:1383-1397; and Wu et al. (1998) Cancer Res. 58:1291-1297. The control of the expression of the human UPII has been studied, and a 3.6-kb region upstream of the mouse UPII gene has been identified which can confer urothelial-specific transcription on heterologous genes (Lin et al. (1995) Proc. Natl. Acad. Sci. USA 92:679-683). See also, U.S. Pat. Nos. 5,824,543 and 6,001,646.
Melanoma, a malignant neoplasm derived from melanocytes of the skin and other sites, has been increasing in incidence worldwide. The American Joint Committee on Cancer recognizes five different forms of extraocular melanoma occurring in humans: lentigo maligna melanoma; radial spreading; nodular; acral lentiginous; and unclassified. Known melanoma-associated antigens can be classified into three main groups: tumor-associated testis-specific antigens MAGE, BAGE, GAGE, and PRAME; melanocyte differentiation antigens tyrosinase, Melan-A/MART-1 (for Melanoma Antigen Recognized by T cells), gp100, tyrosinase related protein-1(TRP-1), tyrosinase related protein-2 (TRP-2); and mutated or aberrantly expressed antigens MUM-1, cyclin-dependent kinase 4 (CDK4), beta-catenin, gp100-in4, p15, and N-acetylglucosaminyltransferase V. See, for example, Kirkin et al. (1998) Exp. Clin. Immunogenet. 15:19-32. Tyrosinase, TRP-1, and TRP-2 are enzymes involved in melanin biosynthesis and are specifically expressed in melanocytes. Antigenic epitopes of MART-1 have been studied extensively, with the aim of developing a melanoma vaccine. An immunodominant epitope, MART-1(27-35) has been reported to be recognized by a majority of CD8+cytotoxic T cell clones generated to MART-1. These MART-1(27-35)-specific CTLs specifically lyse autologous tumor cell lines expressing the epitope. Faure and Kourilsky (1998) Crit. Rev. Immunol. 18:77-86. However, others have reported that presence of such CTLs is not accompanied by a significant clinical response. Rivoltini et al. (1998) Crit. Rev. Immunol. 18:55-63.
A major, indeed the overwhelming, obstacle to cancer therapy is the problem of selectivity; that is, the ability to inhibit the multiplication of tumor cells without affecting the functions of normal cells. For example, in traditional chemotherapy of prostate cancer, the therapeutic ratio, (i.e., the ratio of tumor cell killing to normal cell killing) is only 1.5:1. Thus, more effective treatment methods and pharmaceutical compositions for therapy and prophylaxis of neoplasia are needed.
Accordingly, the development of more specific, targeted forms of cancer therapy, especially for cancers that are difficult to treat successfully, is of particular interest. In contrast to conventional cancer therapies, which result in relatively non-specific and often serious toxicity, more specific treatment modalities, which inhibit or kill malignant cells selectively while leaving healthy cells intact, are required.
Gene therapy, whereby a gene of interest is introduced into a malignant cell, has been attempted as an approach to treatment of many cancers. See, for example, Boulikas (1997) Anticancer Res. 17:1471-1505, for a description of gene therapy for prostate cancer. A gene of interest can encode a protein which is converted into a toxic substance upon treatment with another compound, or it can encode an enzyme that converts a prodrug to a drug. For example, introduction of the herpes simplex virus gene encoding thymidine kinase (HSV-tk) renders cells conditionally sensitive to ganciclovir. Zjilstra et al. (1989) Nature 342: 435; Mansour et al. (1988) Nature 336: 348; Johnson et al. (1989) Science 245: 1234; Adair et al. (1989) Proc. Natl. Acad. Sci. USA 86: 4574; Capecchi (1989) Science 244: 1288. Alternatively, a gene of interest can encode a compound that is directly toxic, such as, for example, diphtheria toxin. To render these treatments specific to cancer cells, the gene of interest is placed under control of a transcriptional regulatory element (TRE) that is specifically (i.e., preferentially) active in the cancer cells. Cell- or tissue-specific expression can be achieved by using a TRE with cell-specific enhancers and/or promoters. See generally Huber et al. (1995) Adv. Drug Delivery Reviews 17:279-292.
A number of viral vectors and non-viral delivery systems (e.g., liposomes), have been developed for gene transfer. Of the viruses proposed for gene transfer, adenoviruses are among the most easily produced and purified. Adenovirus also has the advantage of a high efficiency of transduction (i.e., introduction of the gene of interest into the target cell) and does not require cell proliferation for efficient transduction. In addition, adenovirus can infect a wide variety of cells in vitro and in vivo. For general background references regarding adenovirus and development of adenoviral vector systems, see Graham et al. (1973) Virology 52:456-467; Takiff et al. (1981) Lancet 11:832-834; Berkner et al. (1983) Nucleic Acid Research 11:6003-6020; Graham (1984) EMBO J 3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci. USA 91:8802-8806.
Adenoviruses generally undergo a lytic replication cycle following infection of a host cell. In addition to lysing the infected cell, the replicative process of adenovirus blocks the transport and translation host cell mRNA, thus inhibiting cellular protein synthesis. For a review of adenoviruses and adenovirus replication, see Shenk, T. and Horwitz, M. S., Virology, third edition, Fields, B. N. et al., eds., Raven Press Limited, New York (1996), Chapters 67 and 68, respectively.
When used for gene transfer, adenovirus vectors are often designed to be replication-defective and are thus deliberately engineered to fail to replicate in the target cell. In these vectors, the early adenovirus gene products E1A and/or E1B are often deleted, and the gene to be transduced is commonly inserted into the E1A and/or E1B region of the deleted virus genome. Bett et al. (1994) supra. Such vectors are propagated in packaging cell lines such as the 293 line, which provides E1A and E1B functions in trans. Graham et al. (1987) J. Gen. Virol 36:59-72; Graham (1977) J. Gen. Virol. 68:937-940. The use of replication-defective adenovirus vectors as vehicles for efficient transduction of genes has been described by, inter alia, Stratford-Perricaudet (1990) Human Gene Therapy 1:241-256; Rosenfeld (1991) Science 252:431-434; Wang et al. (1991) Adv. Exp. Med. Biol. 309:61-66; Jaffe et al. (1992) Nature Gen. 1:372-378; Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992) Cell 68:143-155; Stratford-Perricaudet et al. (1992) J. Clin. Invest. 90:626-630; Le Gal Le Salle et al. (1993) Science 259:988-990; Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234; Ragot et al. (1993) Nature 361:647-650; Hayaski et al. (1994) J. Biol. Chem. 269:23872-23875; and Bett et al. (1994) supra.
In the treatment of cancer by replication-defective adenoviruses, the host immune response limits the duration of repeat doses at two levels. First, the capsid proteins of the adenovirus delivery vehicle itself are immunogenic. Second, viral late genes are frequently expressed in transduced cells, eliciting cellular immunity. Thus, the ability to repeatedly administer cytokines, tumor suppressor genes, ribozymes, suicide genes, or genes which convert a prodrug to an active drug has been limited by the immunogenicity of both the gene transfer vehicle and the viral gene products of the transfer vehicle, coupled with the transient nature of gene expression. Despite these limitations, development of adenoviral vectors for gene therapy has focused almost exclusively on the use of the virus as a vehicle for introducing a gene of interest, not as an effector in itself. In fact, replication of adenovirus vectors has been viewed as an undesirable result, largely due to the host immune response.
More recently, however, the use of adenovirus vectors as effectors has been described. International Patent Application Nos. PCT/US98/04080, PCT/US98/04084, PCT/US98/04133, PCT/US98/04132, PCT/US98/16312, PCT/US95/00845, PCT/US96/10838, PCT/EP98/07380 and U.S. Pat. No. 5,998,205. Adenovirus E1A and E1B genes are disclosed in Rao et al. (1992, Proc. Natl. Acad. Sci. USA vol. 89: 7742-7746).
Replication-competent adenovirus vectors, which take advantage of the cytotoxic effects associated with adenovirus replication, have recently been described as agents for effecting selective cell growth inhibition. In such systems, a cell-specific transcriptional regulatory element (TRE) is used to control the expression of a gene essential for viral replication, thus limiting viral replication to cells in which the TRE is functional. See, for example International Patent Application No. PCT/EP99/07380, Henderson et al., U.S. Pat. No. 5,698,443; Hallenbeck et al., PCT/US95/15455 and U.S. Pat. No. 5,998,205; Rodriguez et al. (1997) Cancer Res. 57:2559-2563.
PCT publication PCT/US98/04080 discloses replication-competent, target cell-specific adenovirus vectors comprising heterologous TREs, such as those regulating expression of prostate-specific antigen (PSA), probasin (PB), xcex1-fetoprotein (AFP), kallikrien (hKLK2), mucin (MUC1) and carcinoembryonic antigen (CEA). PCT/US98/04084 discloses replication-competent adenovirus vectors comprising an xcex1-fetoprotein (AFP) TRE that replicate specifically in cells expressing AFP, such as hepatoma cells.
Internal ribosome entry sites (IRES) are sequences which initiate translation from an internal initiation codon (usually AUG) within a bi- or multi-cistronic RNA transcript continuing multiple protein coding regions. IRES have been characterized in encephalomyocarditis virus and related picornaviruses. See, for example, Jackson et al. (1995) RNA 1: 985-1000 and Herman (1989) Trends in Biochemical Sciences 14(6): 219-222. IRES sequences are also detected in mRNAs from other viruses such as cardiovirus, rhinovirus, aphthovirus, hepatitis C virus (HCV), Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). The presence of IRES in cellular RNAs has also been described. Examples of cellular mRNAs containing IRES include those encoding immunoglobulin heavy-chain binding protein (BiP), vascular endothelial growth factor (VEGF), fibroblast growth factor 2, insulin-like growth factor, translational initiation factor eIF4G, and the yeast transcription factors TFIID and HAP4. See, for example; Macejak et al. (1991) Nature 353:90-94; Oh et al. (1992) Genes Dev. 6:1643-1653; Vagner et al. (1995) Mol. Cell. Biol. 15:35-44; He et al. (1996) Proc. Natl. Acad. Sci USA 93:7274-7278; He et al. (1996) Gene 175:121-125; Tomanin et al. (1997) Gene 193:129-140; Gambotto et al. (1999) Cancer Gene Therapy 6:45-53; Qiao et al. (1999) Cancer Gene Therapy 6:373-379. Expression vectors containing IRES elements have been described. See, for example, International Patent Application No. PCT/US98/03699 and International Patent Application No. PCT/EP98/07380.
Thus, there is a continuing need for improved replication-competent adenovirus vectors in which cell-specific replication can be further enhanced, while minimizing the extent of replication in non-target (i.e., non-cancerous cells).
The disclosure of all patents and publications cited herein are incorporated by reference in their entirety.
The present invention provides improved replication competent adenovirus vectors comprising co-transcribed first and second genes under transcriptional control of a heterologous, target cell-specific transcriptional regulatory element (TRE), wherein the second gene is under translational control of an internal ribosome entry site (IRES). In one embodiment, the first and second genes are co-transcribed as a single mRNA and the second gene has a mutation in or deletion of its endogenous promoter. The present invention further provides host cells and methods using the adenovirus vectors.
In one aspect, the first and/or second genes are adenovirus genes and in another aspect, the first and/or second adenovirus genes are essential for viral replication. An essential gene can be an early viral gene, including for example, E1A; E1B; E2; and/or E4, or a late viral gene. In another aspect an early gene is E3.
In one embodiment, the first gene is an adenovirus gene and the second gene is a therapeutic gene. In another embodiment, both genes are adenovirus genes. In an additional embodiment, the first adenovirus gene is E1A, and the second adenovirus gene is E1B. Optionally, the endogenous promoter for one of the co-transcribed adenovirus gene essential for viral replication, such as for example, E1A, is deleted and/or mutated such that the gene is under sole transcriptional control of a target cell-specific TRE.
In another aspect, the present invention provides adenovirus vectors comprising an adenovirus gene essential for viral replication under control of a target cell-specific TRE, wherein said adenovirus gene has a mutation of or deletion in its endogenous promoter. In one embodiment, the adenovirus gene is essential for viral replication. In another embodiment, the adenovirus gene is E1A wherein the E1A promoter is deleted and wherein the E1A gene is under transcriptional control of a heterologous cell-specific TRE. In another embodiment, the adenovirus gene is E1B wherein the E1B promoter is deleted and wherein the E1B gene is under transcriptional control of a heterologous cell-specific TRE.
In another aspect, the present invention provides adenovirus vectors comprising E1B under control of a target cell-specific TRE, wherein said E1B has a deletion in or mutation of the 19-kDa region of E1B, that encodes a product shown to inhibit apoptosis.
In other embodiments, an enhancer element for the first and/or second adenovirus genes is inactivated. The present invention provides an adenovirus vector comprising E1A wherein an E1A enhancer is inactivated. In yet other embodiments, the present invention provides an adenovirus vector comprising E1A wherein the E1A promoter is inactivated and E1A enhancer I is inactivated. In further embodiments, the present invention provides an adenovirus vector comprising a TRE which has its endogenous silencer element inactivated.
Any TRE which directs cell-specific expression can be used in the disclosed vectors. In one embodiment, TREs include, for example, TREs specific for prostate cancer cells, breast cancer cells, hepatoma cells, melanoma cells, bladder cells and/or colon cancer cells. In another embodiment, the TREs include, probasin (PB) TRE; prostate-specific antigen (PSA) TRE; mucin (MUC1) TRE; xcex1-fetoprotein (AFP) TRE; hKLK2 TRE; tyrosinase TRE; human uroplakin II TRE (hUPII) and carcinoembryonic antigen (CEA) TRE. In other embodiments, the target cell-specific TRE is a cell status-specific TRE. In yet other embodiments, the target cell-specific TRE is a tissue specific TRE.
In additional embodiments, the adenovirus vector comprises at least one additional co-transcribed gene under the control of the cell-specific TRE. In another embodiment, an additional co-transcribed gene is under the translational control of an IRES.
In another aspect of the present invention, adenovirus vectors further comprise a transgene such as, for example, a cytotoxic gene. In one embodiment, the transgene is under the transcriptional control of the same TRE as the first gene and second genes and optionally under the translational control of an internal ribosome entry site. In another embodiment, the transgene is under the transcriptional control of a different TRE that is functional in the same cell as the TRE regulating transcription of the first and second genes and optionally under the translational control of an IRES.
The present invention also provides compositions comprising the replication-competent adenovirus vectors described herein. In one embodiment, the compositions further comprise a pharmaceutically acceptable excipient. The present invention also provides kits comprising the replication-competent adnenovirus vectors described herein.
Host cells comprising the disclosed adenovirus vectors are also provided. Host cells include those used for propagation of a vector and those into which a vector is introduced for therapeutic purposes.
In another aspect, methods are provided for propagating replication-competent adenovirus vectors of the present invention specific for mammalian cells which permit the function of a target cell-specific TRE, said method comprising combining an adenovirus vector(s) described herein with mammalian cells that permit the function of a target cell-specific TRE, such that the adenovirus vector(s) enters the cell, whereby said adenovirus is propagated.
In another aspect, methods are provided for conferring selective cytotoxicity in target cells, comprising contacting the cells with an adenovirus vector(s) described herein, whereby the vector enters the cell.
The invention further provides methods of suppressing tumor cell growth, more particularly a target tumor cell, comprising contacting a tumor cell with an adenovirus vector(s) of the invention such that the adenovirus vector enters the tumor cell and exhibits selective cytotoxicity for the tumor cell.
In another aspect, methods are provided for detecting a cell which allows the function of a target cell-specific TRE, which comprise contacting a cell in a biological sample with an adenovirus vector(s) of the invention, and detecting replication of the adenovirus vector(s), if any.
In another aspect, methods are provided for modifying the genotype of a target cell, comprising contacting the cell with an adenovirus vector as described herein, wherein the adenovirus vector enters the cell.
The present invention provides an adenovirus vector comprising an adenovirus gene, wherein said adenovirus gene is under transcriptional control of a melanocyte-specific TRE. In another embodiment, a melanocyte-specific TRE is human. In another embodiment, a melanocyte-specific TRE comprises a melanocyte-specific promoter and a heterologous enhancer. In other embodiments, a melanocyte-specific TRE comprises a melanocyte-specific promoter. In other embodiments, a melanocyte-specific TRE comprises a melanocyte-specific enhancer and a heterologous promoter. In other embodiments, a melanocyte-specific TRE comprises a melanocyte-specific promoter and a melanocyte-specific enhancer.
In some embodiments, the adenovirus gene under transcriptional control of a melanocyte-specific TRE is an adenovirus gene essential for replication. In some embodiments, the adenoviral gene essential for replication is an early gene. In another embodiment, the early gene is E1A. In another embodiment, the early gene is E1B. In yet another embodiment, both E1A and E1B are under transcriptional control of a melanocyte-specific TRE. In further embodiments, the adenovirus gene essential for replication is E1B, and E1B has a deletion in the 19-kDa region.
In some embodiments, the melanocyte-specific TRE is derived from the 5xe2x80x2 flanking region of a tyrosinase gene. In other embodiments, the melanocyte-specific TRE is derived from the 5xe2x80x2 flanking region of a tyrosinase related protein-1 gene. In other embodiments, the melanocyte-specific TRE is derived from the 5xe2x80x2-flanking region of a tyrosinase related protein-2 gene. In other embodiments, the melanocyte-specific TRE is derived from the 5xe2x80x2 flanking region of a MART-1 gene. In other embodiments, the melanocyte-specific TRE is derived from the 5xe2x80x2-flanking region of a gene which is aberrantly expressed in melanomas.
In other embodiments, the invention provides an adenovirus vector comprising (a) an adenovirus gene under transcriptional control of a melanocyte-specific TRE; and (b) an E3 region. In some of these embodiments the E3 region is under transcriptional control of a melanocyte-specific TRE.
In another aspect, the invention provides a host cell comprising the melanocyte specific adenovirus vector(s) described herein.
In another aspect, the invention provides pharmaceutical compositions comprising a melanocyte specific adenovirus vector(s) described herein.
In another aspect, the invention provides kits which contain a melanocyte adenoviral vector(s) described herein.
In another aspect, methods are provided for conferring selective cytotoxicity in target cells (i.e., cells which permit or induce a melanocyte-specific TRE to function), comprising contacting the cells with an adenovirus vector(s) described herein, whereby the vector enters the cell.
In another aspect, methods are provided for propagating an adenovirus specific for melanocytes, said method comprising combining an melanocyte specific adenovirus vector(s) described herein with melanocytes, whereby said adenovirus is propagated.
The invention further provides methods of suppressing melanoma cell growth, comprising contacting a melanoma cell with a melanocyte specific adenoviral vector of the invention such that the adenoviral vector enters the melanoma cell and exhibits selective cytotoxicity for the melanoma cell.
In another aspect, methods are provided for detecting melanocytes, including melanoma cells, in a biological sample, comprising contacting cells of a biological sample with a melanocyte adenovirus vector(s) described herein, and detecting replication of the adenovirus vector, if any.